Na+ efflux rates were also assessedinresponse tohandling stress
using methods similar to Glover et al. (2012). Fish were exposed
to handling stress in a manner identical to that of influx trials,
although rates were measured both as individuals and in groups.
This was to determine any effect of grouping on the stress response,
as inanga are schooling fish (Mitchell, 1989) and therefore may
exhibit altered stress in groups.
For grouped efflux experiments, about 18 fish (total biomass
ranging from 1.7 g L−1 to 3.1 g L−1) were placed in a 10 l plastic
tank (40 cm × 35 cm × 56 cm, n = 3) with approximately 5.5–7 l of
experimental water, with a composition identical to influx trials
(Na+-free, 1 mM Ca2+). Triplicate 5-mL water samples were taken
at time zero, and every hour for the next 3 h, for subsequent measurement
of Na+ concentrations. The same fish were left overnight
to acclimate. After 24 h, fresh aquarium water was added using
partial replacement to gently flush out experimental tanks while
minimising any stress to the fish. Once the overnight water had
been adequately flushed, a new volume of experimental water was
added to the exposure tanks and an identical period of sampling
was undertaken (i.e. water samples taken at time zero and every
hour for the next three hours, 24 h after initial handling).
For efflux experiments with individual fish, a single inanga was
placed in a separate container filled with 250 mL of experimental
water (composition as above). Seven replicate fish were used
(n = 7, mass ranging from 0.7 to 1.4 g) and triplicate 5-mL water
samples taken attime zero, and every hour for the nextthree hours,
in order to determine Na+ concentrations. Fish were left overnight
(24 h) in exposure chambers to acclimate, after which the water
was changed (as described above for the group treatments), by
gently draining and exchanging water in order to minimise additional
stress to the fish.After individual containers were returned to
experimental volumes containing experimental water, 5-mL water
samples were taken at time zero and every hour for the next 3 h for
assessment of Na+ efflux in “unstressed” fish.
During individual fish efflux trials, opercular frequency was also
determined for each of the seven fish for 60 s at the start of each
hour, for a total of four counts per fish. This was done for both
treatments; immediately after handling and 24 h later.